Influenza vaccine

ABSTRACT

The present invention relates to a composition comprising at least one ISCOM complex and at least one ectodomain from at least one hemagglutinin (HA) domain and at least one ectodomain from at least one neuraminidase (NA) domain from one or more influenza virus, wherein the extodomains represent ectodomains isolated from the influenza virus. The invention also regards a kit. The composition may be used as an immune stimulating medicine, immune modulating pharmaceutical or a vaccine e.g. against influenza for vertebrates, e.g. birds and mammals.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.13/811,493, filed Jan. 22, 2013, which is the National Stage ofInternational Application No. PCT/SE2011/050968, filed Jul. 25, 2011,which claims the benefit of priority to U.S. Provisional Application No.61/366,983, filed Jul. 23, 2010, the disclosures of each of which arehereby incorporated by reference in their entireties.

Influenza Vaccine

The present invention relates to a composition comprising at least oneISCOM complex and at least one ectodomain from at least onehemagglutinin (HA) domain and at least one ectodomain from at least oneneuraminidase (NA) domain from one or more influenza virus, wherein theextodomains represent ectodomains isolated from the influenza virus. Theinvention also regards a kit. The composition may be used as an immunestimulating medicine, immune modulating pharmaceutical or a vaccine e.g.against influenza for vertebrates, e.g. birds and mammals.

BACKGROUND ART

The recent emergence of the pandemic swine-origin 2009 A(H1 N1)influenza virus strongly emphasises the potential of influenza virusesto cause morbidity and mortality in the human population on a globalscale. Worldwide over 200 countries and overseas territories orcommunities have reported laboratory-confirmed cases of the pandemicvirus including more than 16,000 deaths [1]. Vaccination is the primarymethod to prevent or lower the burden of influenza disease. However, asillustrated again by the 2009 pandemic, a rapid response during theearly phase of an outbreak is hampered by the time-consuming vaccinestrain preparation and vaccine manufacturing process currently used.This, combined with the notorious capacity of influenza viruses toescape from existing immunity by antigenic drift and shift, stresses theneed for novel, safe and preferably broadly effective vaccines that canbe produced rapidly and in flexible response to newly emerging antigenicvariants.

The currently licensed influenza virus vaccines are composed of theviral envelope glycoproteins, the hemagglutinin (HA) and neuraminidase(NA). Antibodies elicited by these two large glycoproteins have distinctproperties in immunity against influenza virus. Antibodies to HAgenerally neutralise viral infectivity by interference with virusbinding to sialic acid receptors on the target cells or, subsequently,by preventing the fusion of the viral and cellular membrane throughwhich the viral genome gains access to the target cell. Antibodies to NAdisable release of progeny virus from infected cells by inhibiting theNA-associated receptor destroying enzymatic activity. The HA-mediatedhumoral immunity has been characterised most extensively and shown toprevent virus infection. The contribution of NA antibodies to preventingdisease has been studied less well. They appeared to produce a kind ofpermissive immunity [2] characterised by a decrease in infectious virusrelease from apical surfaces of infected epithelia [2-8], reducing theprobability of virus shedding and spread into the environment.

Immunisation with the combination of HA and NA provides enhancedprotection against influenza [5, 9, 10]. Although HA and NA areequivalently immunogenic [2], the humoral immune response towardsconventional inactivated vaccines or virus infection is naturally skewedtowards HA since HA and NA occur on the viral surface at anapproximately 4:1 ratio [11]. In addition, in intact virions the HAimmunologically outcompetes NA in B and T cell priming as shown in mice[12]. This antigenic competition is not seen in vaccinated animals whenHA and NA are administered separately [10, 13]. The currently licensedpandemic vaccines as well as the seasonal trivalent vaccines aregenerally prepared from whole viruses and are hence biased to containmore HA than NA antigen. Adapting the HA-NA ratio in vaccineformulations in favour of NA may provide a more balanced humoral immuneresponse resulting in higher NA antibody levels and increased protectionagainst disease [3, 14].

Since the current inactivated influenza virus vaccines are standardisedonly for the amount of HA, the NA content is variable as is,consequently, the frequency and level of seroconversion to NA, which isoften rather poor [28, 29].

Typical for influenza A viruses, antigenic variants of HA and NA withina certain virus subtype able to escape from existing immunity aregradually selected in the human population. This process of antigenicdrift calls for the almost annual adjustment of the seasonal vaccinecomposition in response to newly arising variants. In view of the threatof future influenza pandemics, caused for instance by an avian H5N1virus, there is a need for vaccines inducing broadly protectiveimmunity.

SUMMARY OF THE INVENTION

The present invention relates to a composition comprising at least oneISCOM complex and at least one ectodomain from at least onehemagglutinin (HA) domain and at least one ectodomain from at least oneneuraminidase (NA) domain from one or more influenza virus, wherein theextodomains represent ectodomains isolated from the influenza virus, anda kit.

Compositions comprising ISCOM adjuvant and HA and NA or fragmentsthereof are known e.g. through WO 2008157419 and [5,9,10]. It hashowever not been disclosed before to use ectodomains from both HA and NAtogether with ISCOM adjuvant.

It has now turned out that vaccination with ectodomains from both NA andHA adjuvanted with ISCOM or ISCOM matrix reduced virus replication—e.g.by lowering pulmonary titers —, and decreased the clinical effects ofinfection such as body weight loss and lung pathology.

Multimeric HA and NA ectodomains have great vaccine potential, as thesecan be easily, rapidly, flexibly and safely produced in high quantities.The inclusion of NA in influenza vaccines, profoundly and specificallycontributes to protection by HA. Its inclusion in a vaccine is likely toreduce the HA dose required and to broaden the protective immunity.

FIGURE LEGENDS

FIG. 1

Design and expression of soluble, multimeric HA (sHA) and NA (sNA)proteins of 2009 A(H1N1) influenza virus. A) Schematic representation ofthe recombinantly expressed sHA and sNA protein constructs. sHA: the HAectodomain (a.a. 17-522) is expressed with an N-terminal CD5 signalpeptide and a C-terminal trimerisation (GCN4-pII) GCN4 domain andStrep-Tag (ST), respectively. sNA: the NA head domain (a.a. 75-469) isexpressed with an N-terminal CD5 signal peptide, a OneSTrEP (OS) peptideand a tetramerisation (GCN4-pLI) GCN4 domain. (B) Coomassie blue stainedreducing SDS-PAGE of affinity-purified sHA and sNA proteins.

FIG. 2

Antibody response to vaccination with multimeric 2009 A(H1N1) influenzavirus HA and NA antigens. Ferrets were immunised on day 0 and day 20with: 3.75 μg sHA₃+3.75 μg sNA₄ (sHA+sNA); 3.75 μg sHA₃ in adjuvant(ISCOM Matrix M [IMM]; sHA+IMM); 3.75 μg sNA₄ in adjuvant (sNA+IMM);3.75 μg sHA₃+3.75 μg sNA₄ in adjuvant (sHA+sNA+IMM), PBS or IMM, asindicated. The antibody response to the 2009 A(H1N1) influenza virus wasevaluated by hemagglutination inhibition (HI; upper panel), virusneutralisation (VN; second panel from the top) and neuraminidaseinhibition (NI) assays (lower panels). Each dot represents the result ofone ferret. Horizontal lines represent means. The horizontal grey barindicates the detection limit of the assay.

FIG. 3

Clinical effects after challenge inoculation with 2009 A(H1N1) influenzavirus. Ferrets immunised as described in the legend to FIG. 2 wereinoculated intratracheally on day 52 with 10⁶ TCID₅₀ of virus. Bodyweight losses are expressed as percentage of body weight beforeinfection (upper panel). Lung weights are expressed as percentage ofbody weight, as an indicator of lung consolidation (middle panel). Lungswere observed macroscopically and scored for lung area percentagedisplaying consolidated areas (bottom panel). Mean values are displayed;error bars indicate standard deviations. The horizontal grey barindicates the detection limit of the assay.

FIG. 4

Examples of histopathologic findings in lungs of ferrets afterinoculation. A) Inflammatory infiltrates and loss of epithelial cells inthe bronchiolar walls and cellular debris in the bronchiolar lumenobserved in the lungs of unprotected ferrets mock-vaccinated with PBS oradjuvant only (IMM) or vaccinated with the non-adjuvanted sHA₃+sNA₄. B)Proteinaceous fluid (edema) and infiltrate of inflammatory cells in thealveoli of lungs of ferrets mock-vaccinated with PBS or adjuvant only(IMM) or vaccinated with the non-adjuvanted sHA₃+sNA₄. C)Peribronchiolar infiltrate and cellular debris in bronchiole of ferretvaccinated with sHA+IMM. D) Inflammatory infiltrate in the alveolarsepta and hypertrophy and hyperplasia of type II pneumocytes in lungs offerrets vaccinated with sHA+IMM. E) Peribronchiolar infiltrate observedin lungs of ferrets of the sNA+IMM and sHA+sNA+IMM groups. F) Absence ofinflammatory cells and hyperplasia of type II pneumocytes in alveoli oflungs of ferrets of the sNA+IMM and sHA+sNA+IMM groups. H&E staining;magnification 20× (bronchioli) and 40× (alveoli).

FIG. 5

Viral titers in lungs, nose and throat of challenge-inoculated animals.Virus replication in the ferrets immunised and challenged as describedin the legend to FIG. 3 was analyzed 4 days after inoculation. Virustiters were determined in lung homogenates (upper panel), nose swaps(middle panel) and throat swaps (bottom panel). Titers were assayed bymeans of end-point titration in MDCK cells. Each dot represents theresult of one ferret. Horizontal lines represent means. The horizontalgrey bar indicates the detection limit of the assay.

FIG. 6

Induction of cross-neutralising antibodies by vaccination withmultimeric 2009 A(H1N1) influenza virus sHA3 and sNA4 antigens. (A) Seraof ferrets immunised twice with sHA₃ or sHA₃+sNA₄, both in adjuvant, asdescribed in the legend to FIG. 2, were tested in an HI assay foractivity towards different influenza viruses includingAI/wine/shope/1/56, A/Italy/1443/76, A/NL/386/86, A/Iowa/15/30,A/NL/25/80, A/NewJersey/8/76, A/PR/8/34 and IVR/148 influenza H1N1. Meantiters are displayed; error bars indicate standard deviation. (B) Seraof ferrets immunised once or twice with sNA₄ or sHA₃+sNA₄, both inadjuvant, were pooled and tested in a NI assay for activity against thesNA₄ of A/Kentucky/UR06-0258/2007(H1N1) and A/turkey/Turkey/1/2005(H5N1)influenza virus. The NA of A/California/04/2009(H1N1) was taken along asa positive control. Positive control sera specific for A/NL/602/09(H1N1)or A/turkey/Turkey/1/2005(H5N1) influenza virus were obtained from aferret infected with these viruses. Average titers of two replicates aredisplayed; error bars indicate standard deviations. The horizontal greybar indicates the detection limit of the assay.

DETAILED DESCRIPTION

The invention regards a composition comprising at least one ISCOMcomplex and at least one ectodomain from at least one HA domain and atleast one ectodomain from at least one NA domain from an influenzavirus, wherein the extodomains represent ectodomains isolated from theinfluenza virus.

The ectodomain is the domain of a membrane protein that extends into theextracellular space. Ectodomains are usually the part of a protein thatinitiates contact with surface which leads to signal transduction. Inthe composition it may act as an antigen and the composition may be usedas a vaccine.

Isolated means that the ectodomain is substantially isolated from otherproteins and from the rest of the NA and HA proteins respectively of theinfluenza virus. Minor rest amino acids may be present. The ectodomainmay be the full ectodomain or a part thereof having the same enzymaticand/or antigenic activity. According to one embodiment the part of theectodomain may be the head domain thereof. Such ectodomains being thefull ectodomain or a part thereof having the same enzymatic and/orantigenic activity may be isolated from influenza virus or syntheticallyproduced. They may be presented separated from each other or linkedtogether.

According one embodiment the ectodomain is a soluble ectodomain or asoluble head domain.

One or more of the ectodomains from at least one hemagglutinin domainand one or more of the ectodomains from at least one neuraminidasedomain from the same or different influenza virus species or strains maybe presented as hybridproteins, which may be recombinant.

Recombinantly produced HA and NA antigens allow the development ofvaccines in which the relative amounts of both antigens can be easilycontrolled. Eukaryotic expression systems, both mammalian and insect,are the preferred platforms for production of such glycoproteins in viewof their better preservation of the proteins' natural antigenicstructure. Hybridproteins may be produced as described in Example 1 andaccording to Genscript http://www.genscript.com/gene_synthesis.html.

The influenza virus may be chosen from sub serotypes of influenza e.g.from HxNy, wherein x is 1-16 and y is 1-9. Thus x may be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 and y may be 1, 2, 3, 4, 5, 6,7, 8, 9. The influenza virus may be from any species such as humans,bird, cattle, e.g. bovine species, swines, sheep, goats. Bird influenzavirus e.g. H₁₋₆ N₁₋₉ or human influenza virus H₁₋₃ N₁₋₂ or anycombination of ectodomains from NA and HA thereof may be used. The NAectodomains and/or the HA ectodomains may be from different species ofinfluenza virus, e.g. of human and bird influenza virus. Thus, one ormore human and bird NA ectodomains may be combined with one or morehuman and bird HA ectodomains, whereby the NA and HA may be of differenttypes of HxNy.

According to one embodiment the influenza virus is an influenza A suchas N1 H 1 virus e.g. chosen from the 1918 H1N1 influenza virus (A/SouthCarolina/1/18) and/or the 2004 H5N1 (A/Vietnam/1203/04) H1N1A/California/07/2009 virus and/or the A/California/04/2009 and/or theA/California/09/2009 and/or A/Kentucky/UR06-0258/2007(H1N1) and/orA/turkey/Turkey/1/2005(H5N1).

One or more ectodomains (multimers) from NA and from HA may be used. Thecomposition may comprise 1-5 ectodomains from at least one hemagglutinindomain and 1-5 head domains from at least one neuaminidase domain.

According to one embodiment the at least one hemagglutinin ectodomain ischosen from the trimeric HA extodomain (a.a. 17-522) of theA/California/09/2009.

According to one embodiment the at least one neuraminidase head domainis chosen from the tetrameric NA head domain (a.a. 75-469) of theA/California/09/2009 virus. The sequence of the A/California/09/2009protein is known in the art.

The ISCOM complexes may be used as adjuvants and immune modulatingagents. They may be ISCOM and/or ISCOM matrix complexes.

The ISCOM-matrix complex and/or the ISCOM complex may be used togetherwith one or more of antigens intended to elicit immune response toinfluenza infection. The antigens and ISCOM-matrix complex and/orISCOM-complex may be administered in mixture or separately.

An ISCOM matrix complex comprises at least one glycoside and at leastone lipid. The glycoside has adjuvant effect and is preferably a saponinespecially form Quillaja saponaria Molina (Quil A). The lipid is atleast a sterol such as cholesterol and optionally also a phospholipid.The ISCOM matrix complexes may also contain one or more otherimmunomodulatory (adjuvant-active) substances, not necessarily aglycoside, and may be produced as described in EP 0 436 620 B1.

An ISCOM complex contains at least one glycoside, at least one lipid,and at least one kind of antigen substance or epitope. These antigensubstances may be of different kind such as proteins and peptides,glycoproteins and glycopeptides, carbohydrates etc. The glycoside hasadjuvant effect and is preferably a saponin especially form Quillajasaponaria Molina (Quil A). These complexes enhance the immunogenicity ofthe included antigens and may also contain one or more immunomodulatory(adjuvant-active) substances. ISCOMs may be prepared as described in EP0 109 942 B1, EP 0 242 380 B1 and EP 0 180 546 B1. Moreover, a transportand/or passenger antigen may be used, as described in EP 9600647-3(PCT/SE97/00289).

The ectodomain antigens may be mixed with ISCOM matrix complex and/orISCOM complex, associated or conjugated on to ISCOM matrix complex ormixed with ISCOM or be coupled onto ISCOM complexes. The prepared ISCOMcomplex may comprise one or more other antigens than the ectodomainantigens. According to one embodiment one or more ectodomain antigensmay be used integrated into ISCOM complex. Such ISCOM complex may thenbe mixed with one or more ectodomain antigens. One or more antigens maybe used and a transport and passenger antigen may be used as describedin EP 9600647-3 (PCT/SE97/00289).

In order to be integrated into ISCOM particles the antigens need to havesome hydrophobic portion or be electrostatic attached to an ISCOMMatirix. Antigens that do not have hydrophobic portions may be coupledto such molecules. Hydrophobic molecules and coupling methods aredescribed in EP 180564.

The lipid comprised in the immunogenic complex is at least a sterol suchas cholesterol and optionally also a phospholipid. The lipid(s) used areparticularly those described in the applicant's patent EP 0 109 942 B1in particular on p. 3 and in patent EP 0 436 620 B1 on p. 7 lines 7-24.Especially sterols such as cholesterol and phospholipids such asphosphatidylethanolamin and phosphatidylcolin are used.

Lipid-containing receptors that bind to the cell-binding components,such as glycolipids including the cholera toxin's receptor, which is theganglioside GM1, and fucosed blood group antigen may be used. Thecell-binding components can then function as mucus targeting moleculeand be bound to the lipid-containing substances through simply mixingthem with complexes that contain them. ISCOM complexes comprisingreceptors are described in e.g. WO 97/30728.

In one embodiment of the invention, the glycoside in the immunogeniccomplex for use in vaccination against influenza is a saponin fractionfrom Quillaja saponaria Molina.

Any sub fragments of Quillaja saponaria Molina saponins may also beused. Moreover, any combination of sub fragments of Quillaja saponariaMolina may be used. Thus, two or more sub fragments may each beintegrated into an ISCOM complex or ISCOM-matrix complex.

The term “a saponin fraction from Quillaja saponaria Molina” is usedthroughout this specification and in the claims as a generic descriptionof a semi-purified or defined saponin fraction of Quillaja saponaria ora substantially pure fraction. It is important that the fraction doesnot contain as much of any other fraction to negatively affect the goodresults that are obtained when the mixtures of ISCOM complexes or ISCOMmatrix complexes comprising essentially one fraction is used.

According to one embodiment, there is provided an immunogenic complexfor use according to the invention, further comprising, as part of theimmunogenic complex or in mixture therewith, at least an additionaladjuvant other than a saponin fraction from Quillaja saponaria Molina orat least one other glucoside or saponin than the one or the onesintegrated into the ISCOM matrix or the ISCOM complex. Examples ofuseful non saponin adjuvants are different oils and Al(OH)₃

Examples of additional adjuvants that can be incorporated in the ISCOMcomplex and ISCOM matrix complex, respectively or mixed therewith, areany adjuvant, natural or synthetic, with desired imunomodulatory effect,e.g. naturally occurring, or derivatives thereof, synthetic or semisynthetic saponin molecules derived from crude saponin extract ofQuillaja saponaria Molina; e.g. saponins and saponin fractions from QuilA, cell wall skeleton, block polymers, e.g. hydrophilic blockcopolymers, e.g. CRL-1005, TDM (threhalose di mucolate), lipopeptides,LPS and LPS-derivatives, Lipid A from various bacterial species andderivatives thereof, (e.g., monophosphoryl lipid A, muramyl di or tripeptide or muramyl dipeptide), MDP-derivatives, (e.g. fatty acidderivatives), substituted MDP, threonyl analogues of MDP(and derivativesthereof), CpG variants, CpGODN variants, endogenous human and animalimmunomodulators, (e.g. GM-CSF). IL-2, adjuvant active bacterial toxins,native or modified toxins, (e.g. cholera toxin CT, and its subcomponentsCTB and CTA1), thermo labile toxin (LT) of E. coli, Bordetella pertussis(BP) toxin and the filamentus heamagglutenin of BP. DDA, poly anionssuch as Dextran sulphate and lipopolysaccarides such as saponins (otherthan Quil A), see(“Future prospects for vaccine adjuvants”, Warren, H.S. (1988) CRC Crit. Rev. Immunol. 8:2, 83-101; “Characterisation of anon-toxic monophosphoryl lipid A”, (1987) Johnson, A. G. et al, Rev.Infect. Dis. 9:5, 5512-5516; “Developmental status of syntheticimmunomodulators”, Berendt, M. J. et al (1985), Year Immunol. 193-201;“Immunopotentiating conjugates”, Stewart-Tull, D. E., Vaccine, 85, 3:1,40-44) all of which are incorporated by reference.

The ISCOM particle may be an ISCOM complex or an ISCOM matrix complexmade from any saponin. The adjuvant fraction A and the other at leastone adjuvant may also be coupled on to the different or the same ISCOMparticles or ISCOM matrix particles or one or more of the adjuvants maybe mixed with the ISCOM particles.

In order to be integrated into ISCOM particles the adjuvants need tohave some hydrophobic molecule. Adjuvants that do not have hydrophobicportions may be coupled to such molecules. Hydrophobic molecules andcoupling methods are described in EP 180564. Preferably the adjuvantsare integrated into different ISCOM particles.

In another embodiment of the invention the adjuvant fraction A of Quil Ais integrated into ISCOM particles, whereas the other at least oneadjuvant are not integrated into ISCOM particles and are used in freeform in the composition.

In another preferred embodiment of the invention the adjuvant fractionsof Quil A is integrated into ISCOM particles or ISCOM matrix particles,whereas other adjuvants are not integrated into ISCOM particles or ISCOMmatrix particles and are used in free form in the composition.

In another especially preferred embodiment the composition comprisesfraction A of Quil A integrated into ISCOM particles or ISCOM matrixparticles and at least one other adjuvant, which is not integrated intoISCOM particles or ISCOM matrix particles.

In another preferred embodiment the at least other adjuvant is MPL orcholera toxin CT. The MPL or cholera toxin may be integrated into thesame ISCOM particle or ISCOM matrix particle or into each a differentISCOM particle or ISCOM matrix particle. Preferably the MPL or choleratoxins are in free form.

The saponin fraction from Quillaja saponaria Molina for use in the ISCOMmatrix complex, the ISCOM complex and/or the at least one additionaladjuvant may be selected from fraction A, fraction B, fraction C ofQuillaja saponaria Molina, a crude fraction of Quillaja saponariaMolina, QA 1-21. However, with modern powerful separation techniquesmore than 60 different structures are reported and described (Bankeforset al., J Chrom B Analyt Technol Biomed Life Sci in press; Bankefors etal., Rapid Commun Mass Spectrom 22:3851; Broberg et al., J Mass Spectrom39:691; Nyberg et al., Anal Chem 75:268; Guo and Kenne Phytochemistry55:419; Nord and Kene Carbohydr Res 329:817; Guo and KennePhytochemistry 54:615; Guo et al., Phytochemistry 53: 861; Nyberg etal., Carbohyd. Res 323:87: Nord and Kenne Carbohyd. Res 320:70 Guo etal., Phytochemistry 48:175).

When prepared as described herein, Fractions A, B and C of Quillajasaponaria Molina each represent groups or families of chemically closelyrelated molecules with definable properties. The chromatographicconditions under which they are obtained are such that thebatch-to-batch reproducibility in terms of elution profile andbiological activity is highly consistent.

The term “one saponin fraction from Quillaja saponaria Molina.” is usedthroughout this specification and in the claims as a generic descriptionof a semi-purified or defined saponin fraction of Quillaja saponaria ora substantially pure fraction. It is important that the fraction doesnot contain as much of any other fraction to negatively affect the goodresults that are obtained when the mixtures of ISCOM or ISCOM matrixcomprising essentially one fraction is used. The saponin preparationmay, if desired, include minor amounts for example up to 40% by weight,such as up to 30% by weight, up to 25% by weight, up to 20% by weight,up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5%by weight, up to 2% by weight, up to 1% by weight, up to 0.5% by weightup to 0.1% by weight of other compounds such as other saponins or otheradjuvant materials.

The saponin fractions A, B and C according to the present invention areas described in WO 96/11711, the B3, B4 and B4b fractions as describedin EP 0 436 620; the fractions QA1-21 are as described in EP 0 3632 279B2. The fractions QA-1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-20and 21 of EP 0 3632 279 B2, especially QA-7, 17-18 and may be used. Theyare obtained as described in EP 0 362 279 B2, especially on page 6 andin Example 1 on page 8 and 9.

Any type of crude or semi-purified fractions of saponins from Quillajasaponaria Molina may be used. A crude fraction of Quillaja saponariaMolina used for the purpose of this patent is any saponin compositionsomewhat purified from other lipophilic non-saponin components. This maybe any saponin fraction which is a purified but non fractionatedpreparation of Quillaja saponaria Molina Such crude fraction wherein thesaponins are not separated from each other may be produced and deliveredfrom e.g., Desert King Chile (www.desertkingchile.cl), Sigma-Aldrich(www.sigmaaldrich.com), Berghausen (www.berghausen.com), BrenntagBiosector (www.brenntag-biosector.com).

Fractions A, B and C described in WO 96/11711 are prepared from thelipophilic fraction obtained on chromatographic separation of the crudeaqueous Quillaja saponaria Molina extract and elution with 70%acetonitrile in water to isolate the lipophilic fraction. Thislipophilic fraction is then separated by semi preparative HPLC withelution using a gradient of from 25% to 60% acetonitrile in acidicwater. The fraction referred to herein as “Fraction A” or “QH-A” is, orcorresponds to, the fraction, which is eluted at approximately 39%acetonitrile. The fraction referred to herein as “Fraction B” or “QH-B”is, or corresponds to, the fraction, which is eluted at approximately47% acetonitrile. The fraction referred to herein as “Fraction C” or“QH-C” is, or corresponds to, the fraction, which is eluated atapproximately 49% acetonitrile.

According to one embodiment a crude fraction of saponins is used.

According to another embodiment a crude fraction of saponins may be usedtogether with any other purified saponin fraction, e.g. the differentsaponin fractions mentioned above.

According to one embodiment of the invention the saponin fraction fromQuillaja saponaria Molina, which is integrated into an ISCOM matrixcomplex or an ISCOM complex, or the at least one additional adjuvant,which also is integrated into the ISCOM or ISCOM matrix complex or mixedtherewith, is selected from fraction A, fraction B, fraction C ofQuillaja saponaria Molina, a semipurified preparation of Quillajasaponaria Molina, a purified preparation of Quillaja saponaria Molina,or any purified sub-fraction e.g., QA 1-21.

ISCOM matrix and/or ISCOM complexes, comprising respectively at leasttwo saponin fraction Quillaja saponaria may be used. Any combinations ofweight % of different saponin fractions may be used. Any combination ofweight % of any two fractions may be used e.g. any weight % of fractionA and any weight % of another fraction, e.g. any crude saponin fractionor fraction C of Quillaja saponaria Molina respectively. The ISCOMmatrix and/or ISCOM complexes may comprise from, 0.1 to 99.9 by weight,5 to 95% by weight, 10 to 90% by weight 15 to 85% by weight, 20 to 80%by weight, 25 to 75% by weight, 30 to 70% by weight, 35 to 65% byweight, 40 to 60% by weight, 45 to 55% by weight, 40 to 60%, by weight,50 to 50% by weight, 55 to 45% by weight, 60 to 40% by weight, 65 to 35%by weight, 70 to 30% by weight, 75 to 25% by weight, 80 to 20% byweight, 85 to 15% by weight, 90 to 10% by weight, 95 to 05% by weight,50 to 99% by weight, 60 to 90% by weight, 70 to 90% by weight, 75 to 85%by weight, of one saponin fraction, e.g. fraction A of Quillajasaponaria Molina and the rest up to 100% in each case of interval ofanother saponin e.g. any crude fraction or any other faction e.g.fraction C of Quillaja saponaria Molina, counted on the sum of theweights of the saponin fractions, of Quillaja saponaria Molina in theISCOM matrix and/or ISCOM complexes.

According to one embodiment, there is provided an ISCOM matrix and/orISCOM complex for use according to the invention, comprising from 5-99%by weight of one fraction, e.g. fraction A of Quillaja saponaria Molinaand the rest up to 100% of weight of another fraction e.g. a crudesaponin fraction or fraction C of Quillaja saponaria Molina counted onthe weight of fraction A and fraction C.

According to another embodiment, there is provided an ISCOM matrixand/or ISCOM complex for use according to the invention, comprising from40% to 99% by weight of one fraction, e.g. fraction A of Quillajasaponaria Molina and from 1% to 60% by weight of another fraction, e.g.a crude saponin fraction or fraction C of Quillaja saponaria Molinacounted on the weight of fraction A and fraction C.

According to yet an embodiment, there is provided an ISCOM matrix and/orISCOM complex for use according to the invention, comprising from 70% to95% by weight of one fraction e.g. fraction A of Quillaja saponariaMolina and from 30% to 5% by weight of another fraction, e.g. a crudesaponin fraction or fraction C of Quillaja saponaria Molina counted onthe weight of fraction A and fraction C.

In one embodiment, there is provided an ISCOM matrix and/or ISCOMcomplex for use according to the invention, wherein the saponin fractionfrom Quillaja saponaria Molina is selected from any one of QA 1-21.

According to one aspect of the invention, there is provided acomposition e.g. a vaccine comprising at least one ISCOM matrix and/orISCOM complex according to the invention together with one or moreectodomain antigens and one or more pharmaceutically acceptable,excipients carriers and/or diluents and/or further adjuvants, for use invaccination against influenza.

Such a composition may comprise one or more different types ofISCOM-matrix complexes particles and/or one or more different types ofISCOM complexes particles, each individual type complex particlescomprising one saponin fraction from Quillaja saponaria Molina, whereinthe saponin fraction in one complex is different from the saponinfraction in the other complex particles. The composition may compriseseveral particles. However, according to one embodiment one particle mayonly comprise one single type of fraction of Quillaja saponaria Molina,

Thus, one type of substantially pure saponin fraction or a crude saponinfraction may be integrated into one ISCOM matrix complex or particle andanother type of substantially pure saponin fraction or a crude saponinfraction may be integrated into another ISCOM matrix complex orparticle. A composition or vaccine may comprise at least two typescomplexes or particles each type having one type of saponins integratedinto physically different particles.

In the compositions, mixtures of ISCOM matrix complex particles and/orISCOM complex particles may be used in which one saponin fractionQuillaja saponaria Molina and another saponin fraction Quillajasaponaria Molina are separately incorporated into different ISCOM matrixcomplex particles and/or ISCOM complex particles. Any combinations ofweight % of the different ISCOM complexes based on their content of onesaponin fraction and any other saponin fraction may be use, e.g.fraction A and another fraction, e.g. any crude saponin fraction orfraction C of Quillaja saponaria Molina respectively may be used. These% figures may be the same as mentioned above regarding possible mixturesof saponin fractions in one and the same ISCOM matrix complex particleand/or ISCOM complex particle, now however in separate ISCOM matrixcomplex particles and/or ISCOM complex particles

In still another embodiment the Quil A fraction A is incorporated intoan ISCOM particle or ISCOM matrix particle and the at least one otheradjuvant is incorporated into each a different ISCOM particle or ISCOMmatrix particle, or one or more other fractions of Quil A or one or moreother adjuvants are incorporated into the same ISCOM or ISCOM matrixparticle but different form the particle into which the Quil A fractionA was incorporated or the other at least one adjuvant is/are in freeform.

In the composition, fraction A may be combined with at least one offractions C and crude saponin fraction of from Quillaja saponaria Molinae.g. fraction Q, in the same or different ISCOM complexes and/orISCOM-matrix complexes.

By combining ISCOM-matrix complexes comprising different fractions ofQuillaja saponaria Molina it is possible to produce compositions thatare less toxic to the animals. Hence, in one embodiment, the compositionfor use according to the invention comprises fraction A in combinationwith at least one of fractions C and Q, in the same or different ISCOMcomplexes and/or ISCOM-matrix complexes.

The composition may further comprise one or more pharmaceuticallyacceptable excipients, carriers and/or diluents and further adjuvants

The composition may comprise at least one other adjuvant than a saponinintegrated into an ISCOM complex particle or an ISCOM matrix particle.This further adjuvant may be a saponin fraction from Quillaja saponariaMolina, which may be bound on to or mixed with the immunogenic ISCOMmatrix complex particle or ISCOM complex particle. It may also beanother type of saponin or any other type of adjuvant which may beintegrated into, bound on to or mixed with the immunogenic ISCOM matrixcomplex particle or ISCOM complex particle.

The composition may be a vaccine.

The term “vaccine” herein refers to a material capable of producing animmune response. A vaccine according to this invention would produceimmunity against influenza.

The compositions may be used prophylactic to prevent infection fromoccurring or may be therapeutic to treat pre-existing infections, or maybe for the production of immunological reagents.

Other antigens than antigens from ectodomains from influenza virus maybe integrated into the ISCOM complex, coupled on to ISCOM complex oronto ISCOM matrix complex or mixed with ISCOM complex or ISCOM matrixcomplex. The invention also regards combination vaccines or combinationveterinary medicines for treatment.

The formulation of compositions according to the invention is well knownto persons skilled in the art. Suitable pharmaceutically acceptablecarriers and/or diluents include any and all conventional solvents,dispersion media, fillers, solid carriers, aqueous solutions, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art, and isdescribed, by way of example, in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofaras any conventional media or agent is incompatible with the activeingredient, use thereof in the pharmaceutical compositions of thepresent invention is contemplated. Supplementary active ingredients canalso be incorporated into the compositions.

According to one other aspect the composition may be used as an immunestimulating medicine, immune modulating pharmaceutical or a vaccine e.g.against influenza for vertebrates, e.g. birds and mammals. The mammalmay be a human, companion animals such as cats, dogs, horses, birds suchas parrots, economical important species such as cattle, e.g. bovinespecies, swines, sheep, goats or ferrets, minks.

The invention also regards a kit comprising at least two compartments,wherein one compartment comprises a composition comprising at least oneISCOM complex and at least one ectodomain from at least onehemagglutinin domain and at least one ectodomain from at least oneneuraminidase domain from one or more influenza virus and the othercompartment comprises instructions for use.

According to another aspect the invention relates to a kit comprising atleast two compartments, wherein one compartment comprises ISCOM complexand/or ISCOM matrix complex and the other compartment comprises and atleast one ectodomain from at least one hemagglutinin domain and at leastone ectodomain from at least one neuraminidase domain.

Applicants have especially addressed the efficacy of recombinantlyproduced HA and NA subunits of the 2009 A(H1N1) influenza virus asvaccines against homotypic influenza virus in a ferret model, withparticular emphasis on the contribution of the NA antigen. Soluble,multimeric forms of the HA and NA antigens of the pandemic H1N1 virushave been expressed in a mammalian expression system. The glycoproteinswere purified by single-step affinity chromatography and subsequentlyferrets were immunised either with one or with both antigens and with orwithout ISCOM Matrix M as an adjuvant. The animals respondedserologically to both antigens, but only when administered with theadjuvant. Interestingly, inclusion of NA in the vaccine enhanced thelevels of HA antibodies and of virus neutralising activity. Significantprotection, as judged particularly from the dramatically (5 log₁₀ units)reduced viral lung titers, was observed upon homologous challenge in theanimals immunised with HA-containing vaccines in combination with ISCOMMatrix M. Interestingly, ISCOM matrix M adjuvanted formulationscontaining NA clearly reduced the clinical effects of infection.

All publications mentioned herein are incorporated herein as reference,to the greatest extent permitted by law.

The invention will now be further described by the followingnon-limiting examples.

EXAMPLES Example 1 Preparation of HA and NA Antigens

Materials and Methods

Influenza A Challenge Virus

Influenza virus A/Netherlands/602/2009 was isolated from the first caseof a laboratory confirmed 2009 A(H1N1) infection in The Netherlands byinoculation of 11-day old embryonated chicken eggs [15]. Virus stocks ofinfluenza virus A/Netherlands/602/2009(H1N1) were prepared by infectingconfluent Madin-Darby Canine Kidney (MCCK) cells. After cytopathologicchanges were complete, culture supernatants were cleared by low speedcentrifugation and stored at −70° C. Infectious virus titers weredetermined in MCCK cells as described previously [16]. All experimentswith these viruses were performed under Bio Safety Level (BSL)-3conditions.

Preparation of HA and NA Antigens

Human codon optimized sequences encoding the soluble hemagglutininectodomain (sHA; a.a. 17-522) and the neuraminidase head domain (sNA;a.a. 75-469) of influenza virus A/California/04/2009(H1N1) weresynthesised (GenScript) and cloned into a derivative of expressionplasmid pS1-lg [17] for expression in HEK293T cells. The HA gene waspreceded by a sequence encoding an N-terminal CD5 signal peptide andfollowed by sequences encoding a C-terminal artificial GCN4trimerisation domain (GCN4-pII) [18] and a Strep-tag for affinitypurification (IBA GmbH) as described recently [19, 20]. The NA gene waspreceded by sequences successively coding for an N-terminal CD5 signalpeptide, a double Strep-tag (OneSTrEP; IBA GmbH) and an artificial GCN4tetramerisation domain (GCN4-pLI) [18].

Production of sHA₃ and sNA₄ Antigens

Constructs were designed to express the trimeric HA ectodomain (a.a.17-522) and the tetrameric NA head domain (a.a. 75-469) of the 2009A(H1N1) influenza virus as pictured in FIG. 1 A. The sHA₃ and sNA₄proteins were produced by expression in HEK293T cells and purified fromthe culture medium by affinity chromatography yielding glycoproteins ofthe expected size (FIG. 1B). Gel filtration analysis indicated thetrimeric and tetrameric oligomeric nature of the HA and NA subunits,respectively (data not shown). The multimeric complexes were alsobiologically active, further confirming their native state, as judged bytheir sialic acid binding (sHA₃; manuscript in preparation) andneuraminidase activity (sNA₄; below).

Example 2. Protein Expression and Purification

HEK293T cells were transfected with the sHA and sNA expression plasmidsusing polyethyleneimine (PEI) in a 1:5 ratio (μg DNA: μg PEI). After a 6h incubation period the transfection medium was replaced by 293 SFM IIexpression medium (Invitrogen) supplemented with sodium bicarbonate (3.7g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter),penicillin (100 units/ml), streptomycin (100 μg/ml), glutaMAX (Gibco),and 1.5% DMSO. Tissue culture supernatants were harvested 5-6 days posttransfection and sHA and sNA proteins were purified from the culturemedium using Strep-Tactin affinity chromatography (IBA GmbH). sHA andsNA protein expression and purification was confirmed by westernblotting using a Strep-Tractin-HRP conjugate (IBA GmbH; data not shown)and SDS-PAGE analysis. Oligomerization of the proteins was determined bygel filtration chromatography and by blue-native-PAGE analysis.Quantification of protein amounts was done using BSA as a reference.

Example 3. Immunisations and Infections

Ferrets

Healthy young adult outbred female ferrets (Mustela putorius furo;between 6 and 12 months old) were purchased from a commercial breeder.The animals were checked for the absence of antibodies againstcirculating seasonal A/H1N1 and A/H3N2 influenza viruses and against theswine-origin influenza A/NL/602/09 virus by hemagglutination inhibitionassay. An independent animal ethics committee approved the experimentalprotocol before the start of the experiment.

The ISCOM matrix M adjuvant was prepared as described in WO2004/004762.75 micrograms of a composition comprising 85% Matrix A and 15% Matrix Cin PBS was added to the antigens.

Immunisations and Infections

Thirty-six seronegative ferrets were divided into six groups of 6ferrets each and vaccinated twice with the following formulations: 3.75μg sHA₃+3.75 μg sNA₄ in phosphate buffered saline (PBS) (group 1); 3.75μg sHA₃ in ISCOM Matrix M (IMM, Isconova, Uppsala, Sweden) (group 2);3.75 μg sNA₄ in IMM (group 3); 3.75 μg sHA₃+3.75 μg sNA₄ in IMM (group4); PBS (group 5); IMM (group 6). Vaccinations were performed with aninterval of 20 days under anesthesia with ketamine in the quadricepsmuscles of the hindleg in a total volume of 1 ml. Ferrets were housed ingroups and received food and water ad libitum. At 32 days after the lastvaccination, the animals were anesthetised with ketamine/medetomidine(reversed with atipamezole), weighed and subsequently challengedintratracheally with 1×10⁶ TCID₅₀ of influenza A/NL/602/09(H1N1) in avolume of 3 ml PBS [21, 22]. Ferrets were subsequently monitored threetimes daily for the development of clinical signs. Before infection andtwo and four days after infection, nose and throat swabs of each ferretwere collected while ferrets were anesthetised with ketamine. Four daysafter inoculation, animals were weighed and subsequently euthanized byexsanguination while under anesthesia with ketamine and medetomidine.Necropsies were performed according to standard procedures. One ferretof group 1 died between the first and second vaccination due to reasonsunrelated to the experiment.

Serology

Serum samples were collected before vaccination, at the day of secondvaccination (day 20) and at the day of challenge (day 52). Sera werestored at −20° C. until use. Sera were tested for the presence ofanti-HA antibodies using a hemagglutination inhibition assay (HI-assay)with 1% turkey erythrocytes and for the presence of virus neutralisingantibodies using a micro virus neutralisation assay (VN-assay) asdescribed previously [23, 24]. Sera were tested for the presence ofantibodies reactive with influenza A/NL/602/09(H1N1). For this purpose,reverse genetics viruses were produced. The titers obtained with theseviruses were comparable to those against the wild-type strains (data notshown). Positive control serum specific for influenza A/NL/602/09(H1N1)was obtained from a ferret infected with this virus [15]. Other H1N1influenza viruses used in the HI-assay were A/Netherlands/386/86(NL/86), A/Netherlands/25/80 (NL/80), A/New Jersey/8/76 (NJ/76),A/Swine/shope/1/56 (Sw/56), A/Italy/1443/76 (It/76), A/Iowa/15/30(10/30), A/Puerto Rico/8/34 (Pr/34) and A/Brisbane/59/07 (IVR-148vaccine strain; IVR/148). Serum samples of ferrets infected with theseviruses were used as a positive control in this assay [25].

Sera were also tested for the presence of neuraminidase inhibiting (NI)antibodies using a previously described fetuin-based assay [26].Briefly, 96-well Nunc MaxiSorp plates were coated overnight at 4° C.with 100 μl of 5 μg/ml fetuin. Sixty-μl volumes of serially dilutedserum samples were incubated for 30 minutes at 37° C. with an equalvolume of sNA₄ containing culture supernatant (prediluted in PBS-Ca/Mg[0.901 mM/0.493 mM] to give a half-maximum OD₄₅₀ of 1.5) after which 100μl of the mixture was added to the fetuin-coated wells. After one hourincubation at 37° C., the plates were washed and neuraminidase activitywas subsequently measured by adding peroxidase-labelled peanutagglutinin (2.5 μg/ml; Sigma), incubating for 1 h at room temperature,washing the plates and adding 100 μl of peroxidase substrate (TMB) toeach well. After 5 minutes, the reaction was stopped by the addition of100 μl of 0.3 M phosphoric acid and OD values were measured at 450 nmusing an ELISA reader (EL-808 [BioTEK]). To test the sera forcross-reactive NI antibodies, sNA₄ expression constructs similar to theones described above for A/California/04/2009(H1N1) were also made forthe head domains of A/Kentucky/UR06-0258/2007(H1N1) (a.a. 75-470) andA/turkey/Turkey/1/2005(H5N1) influenza virus (a.a. 55-449). Seraspecific for influenza A/NL/602/09(H1N1) andA/turkey/Turkey/1/2005(H5N1) obtained from a ferret infected with theseviruses were used as a positive control.

Virus Replication in the Upper and Lower Respiratory Tract

Samples of all lobes of the right lung and of the accessory lobe werecollected of the infected ferrets, snap frozen on dry ice with ethanoland stored at −70° C. until further processing. Lung samples wereweighed and subsequently homogenised with a FastPrep-24 (MP Biomedicals,Eindhoven, The Netherlands) in Hank's balanced salt solution containing0.5% lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 μg/mlstreptomycin, 100 U/ml polymyxin B sulfate, 250 μg/ml gentamycin, and 50U/ml nystatin (ICN Pharmaceuticals, Zoetermeer, The Netherlands) andcentrifuged briefly. Nose and throat swabs were stored directly at −70°C. in the same medium as used to homogenise the lung samples.Quadruplicate 10-fold serial dilutions of throat, nose and lung sampleswere used to infect MDCK cells as described previously [16]. HA activityof the culture supernatants collected 5 days post inoculation was usedas indicator of infection. The titers were calculated according to theSpearman-Karber method and expressed as log TCID₅₀ per gram for lungtissue or per ml for swabs [27].

Histopathology

Four days post inoculation (dpi) with influenza A/NL/602/09 virus,ferrets were euthanized and lungs were observed macroscopically andweighed before samples from the right lungs were collected to determinethe virus titers. Subsequently left lung lobes were inflated with 10%neutral buffered formalin. After fixation and embedding in paraffin,lungs were sectioned at 4 μm and tissue sections were examined bystaining with hematoxylin and eosin (HE).

Statistical Analysis

Significance among animal groups was analysed by one-way ANOVA and Tukeytest subsequent to ANOVA. Differences were considered significant atP<0.05.

Results

Antibody Responses Induced by Immunisation with sHA3 and sNA4

The glycoproteins were tested for their ability to induce protectiveimmunity against homologous virus challenge. Ferrets were immunised atdays 0 and 20 with sHA₃+sNA₄ without adjuvant (sHA+sNA), with sHA₃+sNA₄adjuvanted with ISCOM Matrix M (IMM; sHA+sNA+IMM), or with similarlyadjuvanted sHA₃ (sHA+IMM) or sNA₄ (sNA+IMM). Sera were collected at theday of the second immunisation and pre-challenge (days 20 and 52) andantibody responses were measured by HI and VN assays against thehomologous virus and by NI assay (FIG. 2). No responses with any ofthese assays were observed in control animals vaccinated with PBS orwith adjuvant only, but also not in the animals immunised with thenon-adjuvanted mixture of sHA₃ and sNA₄. In contrast, the immunisationswith adjuvanted sHA₃ (sHA+IMM) induced high HI titers, with a geometricmean titer of 91 at day of challenge. Interestingly, the additionalinclusion of sNA₄ (sHA+sNA+IMM) significantly increased the average HItiters to 468 (p<0.05; one-way ANOVA and Tukey test). Also the NItitrations revealed the adjuvant-dependent induction of NA antibodies,which were low after one (FIG. 2) but strongly boosted after twoimmunisations (FIG. 2). However, in this case no clear augmentation ofthese titers was observed due to the coadministration of sHA₃.Consistent with the observed HI titers, high VN titers were found bothin the sHA+IMM and in the sHA+sNA+IMM vaccinated animals (FIG. 2). Alsohere the co-administration of the sNA₄ antigen with the sHA+sNA+IMMvaccine had resulted in an increase in the mean VN titer, average valuesranging from 1:202 to 1:468 in the sHA+IMM and sHA+sNA+IMM groups,respectively.

Protection Against Clinical Signs after Infection with 2009 a(Ht Nt)Influenza Virus

Vaccinated ferrets were challenged with 10⁶ TCID₅₀ 2009 A(H1N1) 5 weeksafter the second vaccination. From day two after inoculation onwards,clinical signs were observed in inoculated ferrets which includedbreathing difficulties, lethargy, decreased appetite and weight loss. Ingeneral, only mild clinical signs were observed in ferrets of groups 2,3 and 4, while more severe symptoms were observed in ferrets of groups1, 5 and 6. Loss of body weight became obvious in the PBS- andIMM-vaccinated control groups as well as in the non-adjuvanted sHA+sNAvaccine group (FIG. 3). Interestingly, the animals immunised withsHA+IMM showed nearly similar weight losses, while body weights were notsignificantly affected after vaccination with both the sNA₄ containingformulations (groups sNA+IMM and sHA+sNA+IMM). More or lessconsistently, the lung weights of the ferrets determined post mortemshowed a corresponding tendency of adjuvanted sNA₄-vaccinated animalshaving the least disease-related increase due to lung consolidation(FIG. 3).

Gross Pathologic and Histopathologic Findings in the Lungs of Ferrets

Four days after inoculation with influenza virus 2009 A(H1N1) virus, thelungs of the ferrets were examined macroscopically and weighed beforesamples were taken for assessing virus replication and histopathologicalchanges. Dark red and firm consolidated areas were observedmacroscopically in lungs of inoculated ferrets. The percentage ofaffected lung tissue was estimated and varied between groups. Meanpercentages of affected areas in the lungs of about 50% were observed inferrets of groups 1, 5 and 6, while the extent of consolidation was lesspronounced in ferrets of groups 2, 3 and 4, which showed less than 25%of the lung area being affected (FIG. 3). Also the relative lung weightwas lower in these groups compared to that in ferrets of groups 1, 5 and6 (FIG. 3).

Histopathological changes observed at day 4 post inoculation in thelungs of ferrets mock-vaccinated with PBS or adjuvant only (IMM) orvaccinated with the nonadjuvanted sHA₃+sNA₄ were characteristic for amoderate to severe necrotizing broncho-interstitial pneumonia.Multifocally, many neutrophils and macrophages and variable numbers oferythrocytes, edema fluid and fibrin were present in the alveoli of thelungs of these ferrets. In addition, inflammatory infiltrates werepresent in the alveolar septa, in the bronchioles, in the bronchi and inthe walls of bronchi and bronchioli. A dramatic reduction inhistopathological changes was observed in the adjuvanted sNA₄-vaccinatedanimals (sNA+IMM and sHA+sNA+IMM groups), while ferrets immunised withsHA+IMM were partially protected from developing pathology (FIG. 4).

Protection Against Virus Replication in the Upper and Lower RespiratoryTract

To measure the effect of vaccination on the virus replication in therespiratory tract, virus titers were determined in lungs, throat andnose 4 days after inoculation. As shown in FIG. 5 the challenge virusreplicated efficiently in the lungs of the control ferrets (PBS and IMMgroups) and in the animals immunised with the non-adjuvanted mixture ofsHA₃ and sNA₄ (sHA+sNA group), with mean viral titers of approximately10⁷-10⁸ TCID₅₀/gram tissue. These viral loads were reduced by about 5log₁₀ units in the animals immunised with the sHA₃ protein in adjuvant(sHA+IMM group) and in animals co-immunised with sHA₃ and sNA₄ inadjuvant (sHA+sNA+IMM group). Mean viral loads were reduced by 2-3 log₁₀units in animals immunised with adjuvanted sNA₄ antigen (sNA+IMM group).

High viral loads in the nose were observed in the control animals (PBSand IMM groups; FIG. 5) at day 4 after the challenge. Though notstatistically significant due to the large variations in titers withingroups, these viral loads appeared somewhat lower in the animalsimmunised with adjuvanted sHA₃ or adjuvanted sNA₄ or with thenon-adjuvanted sHA₃+sNA₄ combination. The highest reduction of noseviral titers was found in animals immunised with the adjuvantedcombination of sHA₃ and sNA₄ antigens. Viral titers in the throat weregenerally high and not significantly affected by vaccination except inthe animals vaccinated with the adjuvanted combination of sHA₃ and sNA₄.These ferrets did not have detectable titers in the throat.

Cross-Reacting Antibody Responses Induced by Immunisation with sHA₃ andsNA₄

To investigate whether the antibodies induced by the sHA₃ and sNA₄antigens could cross-react with other H1N1 influenza viruses weperformed additional HI and NI assays with the post vaccination sera. Asexpected, the highest HI titers were measured against the homologousvirus while various extents of cross-reactivity were observed with arange of other H1-strains (FIG. 6A). Thus, no cross-reactivity wasdetected for A/Swine/shope/1/56, A/Italy/1443/76, A/Iowa/15/30,A/PR/8/34 and IVR/148, whereas significant cross-reactivity was measuredagainst A/NL/25/80 and A/NewJersey/8/76 and, particularly, againstA/NL/386/86, more or less consistent with the sequence similarities oftheir antigenic domains (see table 1).

TABLE 1 Sequence homology of the antigenic regions within HA ofdifferent H1 N1 strains. % sequence homology HA antigenic domainsGenBank Protein [30] relative to H1N1 strains: Accession number:A/California/04/09 A/California/04/09 ACQ76318.1 100  A/Netherlands/602/09 ACQ45338.1 98.6 A/Swine/shope/1/56 * n.a.A/ltaly/1443/76 * n.a. A/Netherlands/386/86 AAK51350.1 66.2 A/lowa/15/30AAD25303.1 76.1 A/Netherlands/25/80 AAK51352.1 67.6 A/NewJersey/8/76AAA43210.1 78.9 A/PuertoRico/8/34 ACV89502.1 64.8 A/Brisbane/59/07(IVR-148 ADI99532.1 56.3 vaccine strain) * sequence not available; n.a.not applicable

This was the case with the sera from both the sHA3+IMM and thesHA3+sNA4+IMM vaccinated animals. Consistent with the earlier observeddifferences in HI activity against the homologous virus (FIG. 2), thelevels of cross-reactivity were markedly higher with the sera fromferrets immunised with sHA3+sNA4+IMM than with those from sHA3+IMMimmunised animals, confirming again the enhancing effect of the sNAiantigen. HI titers against each strain were detected in control sera offerrets infected with the homologous influenza NH1 N1 virus (data notshown).

To investigate the cross-reactivity of the NA antibodies we producedsNA4 glycoprotein complexes of two other N1 influenza viruses, the humanH1 N1 strain A/Kentucky/UR06-0258/2007 and the avian H5N1 strainA/turkey/Turkey/1/2005. When tested in our NI assay there was a strongneuraminidase inhibiting activity with the pooled sera of the sNA4+IMMand sHA₃+sNA4+IMM immunised animals against the avian H5N1 virus sNA4protein while some inhibition of the seasonal H1 N1 virus sNA4 proteinwas observed (FIG. 6B). Of note, a control serum derived from a H5N1virus infected chicken tested negative against both human H1 N1 virussNA4 proteins. derived from a H5N1 virus infected chicken testednegative against both human H1N1 virus sNA₄ proteins.

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The invention claimed is:
 1. A method comprising administering acomposition to a mammal, said composition comprising at least one ISCOMmatrix and at least one ectodomain from at least one hemagglutinindomain and at least one isolated ectodomain from at least oneneuraminidase domain from one or more influenza virus, wherein theectodomains represent ectodomains being isolated from the influenzavirus, the isolated ectodomains being isolated from the rest of thehemagglutinin and neuraminidase proteins; wherein the ectodomains arepresent in the composition at a ratio of neuraminidase ectodomain tohemagglutinin ectodomain that is greater than that of wild typeinfluenza virus; and wherein the at least one ISCOM matrix comprises atleast one saponin and at least one lipid.
 2. The method of claim 1,wherein one or more of the isolated ectodomains from at least onehemagglutinin domain and one or more of the isolated ectodomains from atleast one neuraminidase domain are presented as hybrid proteinscomprising at least one protein or portion thereof not of influenzavirus.
 3. The method according to claim 1, wherein one or more of theisolated ectodomains from at least one hemagglutinin domain and one ormore of the isolated ectodomains from at least one neuraminidase domainare a head domain.
 4. The method according to claim 1, wherein theinfluenza virus is chosen from sub serotypes of influenza e.g. fromHxNy, wherein x is 1-16 and y is 1-9.
 5. The method according to claim4, wherein the influenza virus is chosen from the group consisting of1918 H1N1 influenza virus (A/South Carolina/1/18), 2004 H5N1(A/Vietnam/1203/04), H1N1 influenza virus (A/California/07/2009), H1N1influenza virus (A/California/04/2009), H1N1 influenza virus(A/California/09/2009), H1N1 influenza virus(A/Kentucky/UR06-0258/2007), and H5N1 influenza virus(A/turkey/Turkey/1/2005).
 6. The method according to claim 1, whereinthe composition comprises 1-5 ectodomains from at least onehemagglutinin domain and 1-5 head domains from at least oneneuraminidase domain.
 7. The method according to claim 6, wherein the atleast one hemagglutinin ectodomain is chosen from the trimeric HAextodomain (a.a.17-522) of the A/California/09/2009.
 8. The methodaccording to claim 6, wherein the at least one neuraminidase head domainis chosen from the tetrameric NA head domain (a.a.75-469) of the H1N1influenza virus (A/California/09/2009).
 9. The method according to claim1, further comprising at least one of an additive, an excipient, and afurther adjuvant.
 10. The method according to claim 1, wherein themammal is selected from the group consisting of a human, a cat, a dog, ahorse, a bird, a cattle, bovine species, a swine, sheep, a goats, aferret, and a mink.
 11. The method of claim 1, wherein the compositionis chosen from a group consisting of an immune stimulating medicine,immune modulating pharmaceutical, and a vaccine against influenza. 12.The method of claim 1, wherein the ISCOM matrix comprises separate ISCOMmatrix particles containing Fraction A and Fraction C.
 13. The method ofclaim 12, wherein the separate ISCOM matrix particles are present in acomposition of about 70% (w/w) Fraction A ISCOM matrix particles andabout 30% (w/w) Fraction C ISCOM matrix particles.
 14. The method ofclaim 12, wherein the separate ISCOM matrix particles are present in acomposition of about 85% (w/w) Fraction A ISCOM matrix particles andabout 15% (w/w) Fraction C ISCOM matrix particles.
 15. The method ofclaim 1, wherein the ISCOM matrix comprises ISCOM matrix particlescomprising a mixture of Fraction A ISCOM matrix and Fraction C ISCOMmatrix.
 16. The method of claim 15, wherein the mixture comprises about70% (w/w) Fraction A ISCOM matrix and about 30% (w/w) Fraction C ISCOMmatrix.
 17. The method of claim 15, wherein the mixture comprises about85% (w/w) Fraction A ISCOM matrix and about 15% (w/w) Fraction C ISCOMmatrix.